Prostate cancer metastasis is a serious sequelae of prostate cancer, associated with increased mortality and reduces treatment effectiveness. Androgen withdrawal therapy, the hallmark of treatment promotes tumor regression. However, treatment benefits wane after an average of 18-24 months, necessitating the use of second-line agents. Chemotherapeutic agents though effective, are not curative and there is an urgent need for novel therapies to be initiated early in the course of the disease. Our laboratory has recently shown that activation of the adenosine A3 receptor (A3AR) subtype inhibits prostate cancer cell proliferation and metastasis in rat and human cell lines of androgen-independent metastatic prostate cancer and in SCID mice injected with the AT6.1 rat metastatic prostate cancer line. The "metastatic phenotype" is mediated in part through the generation of high levels of ROS by these cells via the NADPH oxidase system, which activates signaling pathways, such as nuclear factor (NF)-:B and extracellular signal regulated kinase (ERK) mitogen activated protein (MAP) kinase/activator protein (AP-1) transcription factor. Through these mechanisms, ROS positively regulates the expression of proteins linked to the metastatic phenotype. In this proposal, we will test the hypothesis that activation of the A3AR inhibits the NADPH oxidase activity and thereby suppresses downstream signaling pathways, primarily ERK1/2 MAP kinase and NF-:B and metastatic gene expression regulated by ROS. In a severe combined immunodeficient (SCID) mouse model, we will determine the optimal A3AR treatment protocol to effect maximum inhibition of metastasis. The specific goals are: (1) To determine the optimal treatment parameters for using A3AR agonist to reduce prostate cancer metastasis, focusing on optimal doses and treatment schedules and also determining whether these agonists are effective following establishment of cell metastasis. (2) To determine the mechanism(s) by which the A3AR inhibits ROS generation and motility and metastasis of prostate cancer cells. We will focus on NADPH oxidase and determine the mechanism(s) underlying its activation and subunit expression via the ERK1/2 MAP kinase/AP-1 and NF-:B pathways. Overall, these studies will provide novel information concerning the efficacy of and mechanism underlying the anti-metastatic actions of A3AR agonists in prostate cancer and provide a rational basis for using this class of drug clinically. PUBLIC HEALTH RELEVANCE: Prostate cancer metastasis is a serious consequence of prostate cancer, which if left untreated, could result in death. Several treatment options exist, such as androgen withdrawal therapy and the use of drugs such as ketoconazole and Premarin. In addition, certain platinum-based anti-cancer agents are also effective against the disease but do produce significant side effects such as hearing loss and kidney failure. Overall, such treatments are not curative and there is an urgent need for novel therapies early in the course of the disease. We have shown that a compound which mimics an endogenous chemical in the body, adenosine, can reduce the metastasis of prostate cancer to the lung. However, we do not know how this beneficial action is produced. In addition, we would like to optimize the drug treatment to produce more reductions in lung metastasis. Therefore, these goals will form the specific aims of the current grant proposal. We believe that this study could uncover a novel method of treating prostate cancer metastasis. [unreadable] [unreadable] [unreadable]